Localizing Proton-Mediated Inhibitory Feedback at the Retinal Horizontal Cell-Cone Synapse with Genetically-Encoded pH Probes.

Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca2+ channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENT Lateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC-cone synapse is a "private-line" that operates independently from other HC-cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.

Flow of energy in the outer retina in darkness and in light.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Controlling Horizontal Cell-Mediated Lateral Inhibition in Transgenic Zebrafish Retina with Chemogenetic Tools.

Horizontal cells (HCs) form reciprocal synapses with rod and cone photoreceptors, an arrangement that underlies lateral inhibition in the retina. HCs send negative and positive feedback signals to photoreceptors, but how HCs initiate these signals remains unclear. Unfortunately, because HCs have no unique neurotransmitter receptors, there are no pharmacological treatments for perturbing membrane potential specifically in HCs. Here we use transgenic zebrafish whose HCs express alien receptors, enabling cell-type-specific control by cognate alien agonists. To depolarize HCs, we used the Phe-Met-Arg-Phe-amide (FMRFamide)-gated Na+ channel (FaNaC) activated by the invertebrate neuropeptide FMRFamide. To hyperpolarize HCs we used a pharmacologically selective actuator module (PSAM)-glycine receptor (GlyR), an engineered Cl- selective channel activated by a synthetic agonist. Expression of FaNaC or PSAM-GlyR was restricted to HCs with the cell-type selective promoter for connexin-55.5. We assessed HC-feedback control of photoreceptor synapses in three ways. First, we measured presynaptic exocytosis from photoreceptor terminals using the fluorescent dye FM1-43. Second, we measured the electroretinogram (ERG) b-wave, a signal generated by postsynaptic responses. Third, we used Ca2+ imaging in retinal ganglion cells (RGCs) expressing the Ca2+ indicator GCaMP6. Addition of FMRFamide significantly decreased FM1-43 destaining in darkness, whereas the addition of PSAM-GlyR significantly increased it. However, both agonists decreased the light-elicited ERG b-wave and eliminated surround inhibition of the Ca2+ response of RGCs. Taken together, our findings show that chemogenetic tools can selectively manipulate negative feedback from HCs, providing a platform for understanding its mechanism and helping to elucidate its functional roles in visual information processing at a succession of downstream stages.

Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina.

The reciprocal synapse between photoreceptors and horizontal cells underlies lateral inhibition and establishes the antagonistic center-surround receptive fields of retinal neurons to enhance visual contrast. Despite decades of study, the signal mediating the negative feedback from horizontal cells to cones has remained under debate because the small, invaginated synaptic cleft has precluded measurement. Using zebrafish retinas, we show that light elicits a change in synaptic proton concentration with the correct magnitude, kinetics and spatial dependence to account for lateral inhibition. Light, which hyperpolarizes horizontal cells, causes synaptic alkalinization, whereas activating an exogenously expressed ligand-gated Na(+) channel, which depolarizes horizontal cells, causes synaptic acidification. Whereas acidification was prevented by blocking a proton pump, re-alkalinization was prevented by blocking proton-permeant ion channels, suggesting that distinct mechanisms underlie proton efflux and influx. These findings reveal that protons mediate lateral inhibition in the retina, raising the possibility that protons are unrecognized retrograde messengers elsewhere in the nervous system.

Synaptojanin 1 is required for endolysosomal trafficking of synaptic proteins in cone photoreceptor inner segments.

Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1) is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, nrc(a14) , which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of nrc(a14) cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the nrc(a14) inner segment. The structure of the Endoplasmic Reticulum in nrc(a14) mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within nrc(a14) cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of nrc (a14) mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.

Differential role for synaptojanin 1 in rod and cone photoreceptors.

Synaptojanin 1 (SynJ1) is a polyphosphoinositide phosphatase involved in clathrin-mediated endocytosis in conventional synapses. Studies with the zebrafish mutant nrc have revealed that loss of SynJ1 also affects cone photoreceptor ribbon synapses, causing pronounced morphological and functional abnormalities. In this study we continue to examine the role of SynJ1 in photoreceptors. Using a newly generated antibody specific for zebrafish SynJ1, we localized this protein predominantly to cone photoreceptors. We then used blastula stage transplantation experiments to demonstrate that rods from nrc mutants lacking SynJ1 develop normally and do not have the pronounced morphological defects detected in cones. Given the known involvement of SynJ1 in synaptic vesicle endocytosis, we hypothesize that rods and cones use distinct mechanisms for vesicle recycling.

The NH2-terminal propeptide of type I procollagen acts intracellularly to modulate cell function.

The function of the NH(2)-terminal propeptide of type I procollagen (N-propeptide) is poorly understood. We now show that a recombinant trimeric N-propeptide interacts with transforming growth factor-beta1 and BMP2 and exhibits functional effects in stably transfected cells. The synthesis of N-propeptide by COS-7 cells results in an increase in phosphorylation of Akt and Smad3 and is associated with a marked reduction in type I procollagen synthesis and impairment in adhesion. In C2C12 cells, N-propeptide inhibits the osteoblastic differentiation induced by BMP2. Our data suggest that these effects are mediated by the interaction of N-propeptide with an intracellular receptor in the secretory pathway, because they are not observed when recombinant N-propeptide is added to the culture medium of either COS-7 or C2C12 cells. Both the binding of N-propeptide to cytokines and its functional properties are entirely dependent on the exon 2-encoded globular domain, and a mutation that substitutes a serine for a highly conserved cysteine in exon 2 abolishes its function. Our findings suggest that N-propeptide performs an important feedback regulatory function and provides a rationale for the prominence of a homotrimeric form of type I procollagen (alpha1 trimer) during vertebrate development.